UNIVERSITY OF MEDICINE AND PHARMACY OF

CRAIOVA

Implication of epithelial and mesenchymal interrelation in the

evaluation of chronic kidney disease

Clinical, histological and immunohistochemical study

ABSTRACT

Ph.D. Thesis Coordinator

Prof. Univ. Dr. Laurenţiu MOGOANTĂ

Ph.D. Student

Ofelia Claudia JERCAN

CRAIOVA

2013

CONTENT

I. STATE OF KNOWLEDGE ................................................................................................3

I.1. Introduction..................................................................................................................3

I.2. Epidemiology of chronic kidney disease .......................................................................4

I.3. Histopathological characteristics in chronic kidney disease evolution ...........................5

II. PERSONAL CONTRIBUTION.........................................................................................8

II.1. CLINICAL STUDY....................................................................................................8

II.1.1. Matherials and methods........................................................................................8

II.1.2. Results..................................................................................................................9

II.1.3. Discussions.........................................................................................................11

II.2. HISTOLOGICAL STUDY........................................................................................12

II.2.1. Matherials and methods......................................................................................12

II.2.2. Results................................................................................................................13

II.2.3. Discussions.........................................................................................................14

II.3. IMMUNOHISTOCHEMICAL STUDY....................................................................16

II.3.1. Matherials and methods......................................................................................16

II.3.2. Results................................................................................................................18

II.3.3. Discussions.........................................................................................................20

IV.CONCLUSIONS.............................................................................................................22

V.REFERENCES.................................................................................................................24

I. STATE OF KNOWLEDGE

I.1. Introduction

In many epidemiological studies is highlighted the importance of diagnosis of renal

impairment in early stages of chronic kidney disease, the etiology diagnosis being difficult

based only on clinical criteria [41-43].

Thus, various studies support that renal biopsy is essential to obtain a conclusive

diagnosis regarding the etiology of renal impairment, clinical suspicion being confirmed by

renal biopsy in only 51% of patients [44].

However, it is noted that, in patients with advanced chronic kidney disease in renal biopsy

procedure has a higher risk of complications and histopathological diagnosis results in a

modification based therapy [45].

Multiple studies have demonstrated the presence of non-reversible healing mechanisms

hold a key pathogenic factor in the development of chronic kidney disease regardless of initial

histological lesions [92]. Thus, chronic injury to the parenchyma, similar to well differentiated

tissue structures replacement with fibrosis in the healing process, are specific pathogenetic

mechanisms in the development of chronic parenchymal insufficiency in different tissues such

as liver, kidneys and lungs [93].

The purpose of this study was to demonstrate the relationship between epithelial

phenotype and mezemchymal expression in renal interstitial fibrosis development process, as

described, as the expression of new immunohistochemical markers predictive for renal

function and in development and interaction between the expression of these markers and

treatment with vitamin D or vitamin D substitutes.

I.2. Epidemiology of chronic kidney disease

The term chronic kidney disease was introduced in 2002 by the working group "The

Kidney Disease Outcome Quality Initiative" (K / DOQI) and is defined as decreased

glomerular filtration rate (GFR) in 60ml/min/1.73 sqm. body surface , persisting more than 3

months, with / without renal damage or renal impairment as the presence of more than 3

months, demonstrated by pathological changes or the presence of injury markers such as

albuminuria / proteinuria, urinary sediment and kidney changes detectable pathological

imaging [6].

In recent years the term of chronic renal failure was replaced with chronic kidney disease

because it highlights the existence of chronic renal disease in patients with normal GFR (> 90

ml/min/1.73mp- BCR stage I) or slightly reduced from 60 to 90 ml/min/1.73mp or stage II

chronic kidney disease and because is including renal transplant patients [6].

Chronic kidhey disease (CKD) is a major public health problem worldwide because of

higher prevalence of 18.3% in Australia (AUSDIAB) [7] and 11% in the U.S. (NHANES III)

[8]. In Western Europe the prevalence of CKD stages 3-5 is about 10% of the adult population

[6]. In 2004, there were 1,783,000 people of patients in renal replacement therapies whith

1,371,000 (77%) patients on dialysis and 412,000 (23%) patients with transplanted kidney.

In Romania there is an increase in the number of dialysis patients (5447 in 2003) and

roughly double in 2009 and increasing age of initiation of renal function sustitution therapy.

Multiple studies support that renal transplantation improves life expectancy by about 3-15

years compared with renal replacement therapy by dialysis but consistent with the presence of

various features for donors and recipients such as age at transplantation [5] .

Although renal transplantation is the best choice for patients with chronic kidney disease

due to improved quality of life compared to dialysis, there is a problem identified as the

global demand for kidney transplants far exceeds the actual possibilities to achieve it [10 ].

Regarding the survival rate at 1 year for kidney transplant was as follows: 93.7%

probability of survival at 1 year after transplantation and cadaver donor transplantation for life

supraviţuire rate is 97, 6% (Annual Report of USRDS data 2003). 5-year survival rate

presented in the same report for the afost kidney transplant: 80.6% probability of survival at 5

years after transplantation from cadaver and 90.4% probability for a living donor.

In Romania from 1980 until June 1997 were performed 45 kidney transplants, and from

June 1997 to December 2001 were performed 255 kidney transplants in which the donor graft

39 brain dead. In the Annual Report of 2011 of the Romanian Renal Registry has been

reported that although growth in the number of patients with renal graft function is higher

than the European average, the percentage of renal transplantation among renal replacement

therapy methods is still small, so hemodialysis remains the primary method of renal

replacement therapy in Romania (88%) [11].

I.3. Histopathological characteristics in chronic kidney disease evolution

In the kidney, the interstitial fibrosis was considered a common mechanism of progression

of chronic kidney disease, which at some point becomes independent of the original cause

kidney damage and is linked to a pathogenic imbalance between submission and degradation

of extracellular matrix components [13]. This process is stimulated by a variety of cytokines

and growth factors.

Growth factors belonging to the TGF-β family are the most intensively studied molecules

derived from macrophages that are related to renal fibrosis [14].

Macrophages, tubular epithelial cells, and myofibroblasts are able to synthesize TGF-β in

different stages of renal fibrotic lesions. Thus, the observation that suppression of expression

of macrophages in the chronic lesion progression to fibrosis suggests that these cells are

among the main producers of TGF-β [15].

Once activated this factor, TGF-β, signals through SMAD proteins transmembrane

receptor that activates transcription of genes that will regulate important processes as those

coding for collagen synthesis. In the kidney, TGF-β derived from macrophages may promote

fibrosis by activating paracrine expression of extracellular matrix and myofibroblasts

synthesis and also promote tubular epithelial cells trasdiferentiatign in myofibroblasts [16].

Thus, initiation of epithelial- mesenchymal transdifferentiation is an evolving process

evoluating to decline in renal function and renal replacement therapy initiation. In addition,

macrophages can synthesize and secrete collagen and TGF-β can also turn off, macrophages

and induce expression of the macrophage inflammatory phenotype characterized by the

production of collagen type VI [17].

In a recent study, it was observed that a single hematopoietic cell was able to differentiate

into mesangial cells in an experimental model of hematopoietic cell transplantation, thus these

observations demonstrates that these cells derived from bone marrow can rebuild mesangial

and interstitial cells [18].

In a recent study has observed that GFP + bone marrow cells migrate to the glomeruli and

interstitium thus contributes to normal cellular bone turnover [19].

Although the majority of regenerated tubular epithelial cells derived from intrarenal

source of asmenea, bone marrow-derived cells can contribute to replacing tubular epithelial

cells through a process of cell fusion [17].

Thus, hematopoietic cell responsible for cell fusion process is not fully known, although

there is evidence that macrophages can be reponsabilă of this process in the liver [18]. Thus,

despite adequate stocks of cells and mechanisms for repair of damaged, in most cases, the

kidneys or liver fibroblasts are used in the development process of fibrosis [16]. For example,

if glomerular epithelial cells are the only non regenerating line of cells, endothelial cells,

mesangial or tubular cells have high proliferation index providing adequate structural

elements in the healing processes.

One factor that seems to be acting in tubular epithelial cells is represented by angiotensin

II, which acts as a growth factor thereby stimulating local, cellular hypertrophy and

production of collagen type IV and the main growth factors involved in this process is the

TGF β1 [13]. Thus, this mechanism could explain the decrease in TGF-β1 in urine samples

after administration of corticosteroids or inhibitors of angiotensin converting enzyme (ACE).

However, combination therapy with ACE inhibitors and angiotensin II receptor blockers,

significantly enhances this phenomenon. The main factors that trigger TGF-β1 secretion are

angiotensin II and proteinuria. Thus, in patients with diabetic nephropathy and proteinuria

high levels of TGF-β1 in the urine compared to patients without proteinuria [20].

The most important mechanisms involved in chronic injury at tubulo-interstitial level in

nephropathies evolving with proteinuria are: direct injury to tubular epithelial cells by

consumtion of large amounts of protein through phagocytosis and accumulated in excess,

tubular cells ischemia is a phenomenon triggered by multiple chemokines and growth factors

that reach tubular cells, new antigens expression of adhesion molecules and proinflammatory

features tubular cells after attaching to cell surface proteins as well as proteinuria and hypoxia

[22].

TGFβ-1 by SMAD pathway is considered as the main mediator in regulating mechanisms

of epithelial to mesenchymal transition (EMT), together with the factors likely inducers of

hypoxia. Fibroblasts cells are pivotal effectors of fibrogenesis, initially these cells were

identified by light microscopy and electron microscopy based on the characteristics

mofologice but more recently are identified by fibroblast markers. In kidney, Bowman

identified two distinct cell populations in the cortex and the medulla internal morphological

and functional changes in the future in accordance with stage of disease [22].

Given the complexity of cell lines, fibroblasts renal origin remains controversial, currently

the most common theory prioritizes local membership interstitial cells but other authors claim

that migrated leukocytes derived to local fibroblasts [13]. In recent studies performed in

cultured cells and in experimental models of nephropathy has been proposed that tubular

epithelial cells through EMT become collagen-producing cells. According to this hypothesis

epithelial cells should undergo several stages such as proliferation and phenotypic changes to

eventually synthesize extracellular matrix proteins.

Presence of EMT in renal fibrosis was first demonstrated by Strutz et al. [23], using

fibroblast-specific protein (Fsp1) as a marker, these authors showed that tubular epithelial

cells could express Fsp1, calcium binding proteins associated with the cytoskeleton, which is

normally expressed in the fibroblasts.

In obstructive nephropathy induced by unilateral ureteral obstruction, multiple cells

showed co-expression of both α-SMA and tubular markers, indicating that they are in a

transitional stage between epithelium and mezenchyme [25]. These tubular epithelial cells lost

their specific epithelial markers and gained mezemchymal features such as α-SMA and

vimentin, resulting in production of interstitial matrix components such as fibronectin and

collagen type I. According to studies EMT was also observed in tissue obtained by renal

biopsy [2].

Recent studies on biopsy specimens provide new arguments in support of EMT that seems

to play a role in progressive renal fibrosis, tubular epithelial cells that undergo phenotypic

changes, as demonstrated by the expression of α-SMA and cytokeratines loss [24]. Because

EMT often occurs in areas with severe structural tubular damage, a concern that has been

raised over the years is that the α-SMA positive tubular cells may represent an interstitial

infiltration of miofibroblaste.

In conclusion, renal tubulo-interstitial fibrosis characterized by depositing excess tissue in

the renal parenchyma is an extensive process that finally results in progressive renal function

impairement, independent of the primary renal disease.

Hepatocyte growth factor (HGF) and BMF-7 is the TEM molecule inhibitors for which

experimental and clinical puteapreveni at interstitial fibrosis process. Recent studies indicate

that administration of these molecules prevents peritoneal fibrosis in experimental models

[23].

II. PERSONAL CONTRIBUTION

II.1. CLINICAL STUDY

Clinico-statistical study objectives were represented by: description of demographic

characteristics for the study group and determining population distribution by sex, age and

risk factors in chronic kidney disease, evaluation of the onset and confounding pathologies in

chronic kidney disease onset, clinical form of the disease, investigating the factors involved in

the development of renal pathology.

II.1.1. Matherials and methods

The clinical study was conducted in Nefrology Hospital "Carol Davila", and represents a

retrospective study on a group of 87 patients who underwent renal biopsy procedures from

2008 to 2011 and followed up for a period of 21 ± 11 months or until renal function decline

and initiate dialysis or death.

The protocol was approved by the Ethics Committee of the Hospital "Carol Davila" and

was conducted in accordance with the ethical principles of the Helsinki Convention.

Information on clinical and laboratory parameters were extracted from electronic

databases and medical records of each patient. Collection and reporting of these data was

approved by the ethics committee of the institution and informed consent for the use of

confidential data was obtained from each patient.

Exclusion criteria were represented by: lack of clinical and laboratory data at the time of

biopsy and follow-up, lack of tissue in the renal biopsy fragments viable to perform classical

histology procedures.

Renal function was assessed by serum creatinine levels measured at frequent intervals

and by estimating glomerular filtration rate (GFR) by MDRD formula [26].

Basal medical history and clinical parameters were recorded at the time of renal biopsy

and biochemical and clinical reassessment was performed at intervals determined for each

patient according to the recommendations of "Kidney Disease Outcomes Quality Initiative"

for assessing chronic kidney disease [27].

Induction immunosuppressive therapy for primary glomerulonephritis was the cortisol

associated with cyclophosphamide. Immunosuppression in some primary glomerulonephritis

consisted of the combination of prednisone and mycophenolate mofetil.

Renal biopsies were performed under ultrasound guidance by puncture with a needle

mounted in an automatic spring, the main indications for biopsy were: isolated proteinuria,

decreased renal function assessed by serum creatinine increase over 25% from previous

values, the association between decreased kidney function and proteinuria.

Biological samples were obtained from the Department of Nephrology each patient fasted

for 12 hours and all biochemical analyzes were performed in the same laboratory.

Systemic hypertension was defined as blood pressure values above the limit of

consecutive repeated 140/90mmHg or use of at least one anti-hypertensive drug.

Regarding statistical analysis, continuous variables were expressed as mean values ± SD.

Differences between groups were determined by Student t test, Mann-Whitney test and

ANOVA, where indicated. Linear regression and logistic regression were used to perform

single and multi-variable analysis. In all analyzes statistical significance was established for P

values <0.05. Statistical analysis was performed using SPSS software version 9.

II.1.2. Results

In the clinical study were evaluated 87 patients who underwent renal biopsy procedures

between April 2008 - September 2011 and followed up for 21 ± 11 months or until the decline

of renal function and initiation of dialysis.

During the study period a total of 8 (9%) patients had renal function decline with renal

sustitution therapy initiation.

Regarding chronic kidney disease stages it was observed a constant ratio between the

incidence of glomerular filtration rate between 70 and 90ml/min/1.73m ², in the cases in

which there has been observed the loss of renal function it was observed an increased

incidence of cases with a glomerular filtration rate less than 60 ml/min/1.73m at renal biopsy

moment.

For the group of patients who had renal function decline, with the initiation of renal

replacement therapy, the average time to the loss of renal function was 15 ± 10 months.

Study on gender distribution of the studied population showed male predominance in both

groups, so there is a percentage of 61.3% of male patients in the group of patients in whom

there was no decline in renal function. In the stratification of patients in terms of age there

was no difference between the two groups, the average age of the study group was 44.7 ± 11

years.

Following distribution by age, a maximum incidence of patients with eGFR>

90ml/min/1.73m ² was observed in the group aged 30-40 years and also can be observed the

continuing decline in the incidence of case with GFR within 60-90 ml/min/1.73m ² with renal

biopsy indication, in age groups over the 50 years.

As the incidence of eGFR decline in the age groups, there was observed an increasing

trend in the frequency of cases in which there was observed a decreased kidney function in

the age group between 40-49 years and the age group 50-59 years.

Clinical and laboratory criteria that represent the indication to perform renal biopsy in the

group with normal glomerular function, with eGFR calculated by the MDRD formula>

90ml/min/1.73m ², co-existence of proteinuria/24 h> 1g was in 87% of cases the main reason

to perform renal biopsy in association with the presence of micro or macrohematuriei.

Isolated proteinuria with less than 1g/24h, did not influence the decision to perform renal

puncture under normal renal function, even with modified values of serum proteins.

Regarding the presence of cardiovascular risk factors was found that the group who had

renal function decline presented in 62.5% of cases high blood pressure values and in 34% of

cases were associated with more than 3 anti- hypertensive medication.

It was observed in this study the presence of inflammatory syndrome in 75% of cases

where there has been observed the decline in renal function and serum levels of C-reactive

protein at the time of renal biopsy related with glomerular filtration rate (eGFR) during

follow-up (r = -0.21, p = 0.021).

To determine the impact of several factors on renal function decline and initiation of renal

replacement therapy we applied logistic regression statistical model.

The strongest predictor of initiation of substitutive renal therapy, in this model, was serum

creatinine at the time of renal biopsy. Thus, any unit decrease in glomerular filtration rate

increases during follow-up of 1.46 times the risk of starting renal replacement therapy by

dialysis.

Short time tracking is also a predictor of starting dialysis with a probability rate of 1.16,

this means that each month tracking further reduce risk of 1.16 times.

Also, the association of several cardiovascular risk factors relate to decreased glomerular

filtration rate, as was observed that in 75% of cases where there has been obseved the decline

in renal function has been emphasized the biological inflammatory syndrome during follow-

up and in 62.5% of cases initiated renal replacement therapy were evolving metabolic acidosis

which was corrected treated during follow-up.

II.1.3. Discussions

In the clinical-statistical study it was investigated a group with different etiology of renal

impairment and was observed that performing renal biopsy puncture at a greater glomerular

filtration rate was associated with improved survival, respectively, fewer cases had initiated

renal replacement therapy.

Preserved renal function at the time of renal biopsy puncture was an important predictive

factor for chronic kidney disease progression. Thus, immunosuppressive therapy or

modification of this therapy after renal biopsy puncture resulted in improved renal function in

most cases of glomerulonephritis. The response to treatment is comparable to existing data in

other studies in groups of subjects with pathological diagnosis of glomerulonephritis in

different stages [28-31].

This study did not compare individual immunosuppressive therapy in groups of patients

with different etiology of glomerular damage, these subgroups were too small for such an

analysis.

In terms of population distribution by gender was observed in this study, an increased

incidence of males in certain age groups. This observation is supported by a number of studies

that emphasizes higher life expectancy for women [48].

Thus, Haas et al. reported an almost equal number of women and men aged over 80 years

who underwent renal biopsy procedures for the investigation of acute kidney injury (AKI)

[49].

In our study, there was a similar incidence of cases diagnosed with membranous

nephropathy and primary focal segmental glomerulosclerosis in the age group over 50 years.

In the age groups below 50 years it was seen a high incidence of cases diagnosed with

diabetic nephrosclerosis and glomerulopathy with minimal damage.

An important aspect described in this study is represented by underlining the importance

of cardiovascular risk factors in the decline of renal function.

Thus, biological inflammatory syndrome with elevated CRP levels and hypertension were

risk factors associated with decline in glomerular filtration rate (p = 0.001 and p = 0.032).

In analyzing the factors involved in the development of renal pathology an important

aspect was the predictive power of serum creatinine at the renal biopsy moment (p = 0.48),

and decreased glomerular filtration rate during follow-up was a predictive factor for the

initiation of renal replacement therapy .

II.2. HISTOLOGICAL STUDY

Histological study objectives were represented by: histopathological description of the

main features in the study group, highlighting the presence of inflammatory features at

interstitial and tubular expression and describing interstitial fibrosis score and periglomerular

fibrosis in the development of renal pathology.

II.2.1. Matherials and methods

Histological study was performed on a total of 87 pieces of renal biopsy obtained from

patients who underwent renal biopsy procedures between April 2008 - September 2011 and

followed for 21 ± 11 months or until the initiation of dialysis. There were selected only cases

analyzed by microscopy in Nepropathology Laboratory of the Hospital of Nephrology "Carol

Davila", Bucharest.

Microscopic aspects were studied in the tubulo-interstitial and glomerular area and the

degree of inflammation and fibrosis. There were selected renal biopsies that had sufficient

tissue for the diagnosis of all three techniques: optical microscopy, electronic miroscopie

direct imunonofluorescenţă.

For the study histology were used to quantify the histological changes at least three

sectiones in the usual histological stains for each biopsy, a section with hematoxylin-eosin

staining, two sections with Goldner-Szeckeli trichrome staining, all sections had more than 5

glomeruli. In all biopsies for histological diagnosis there were reviewed by two independent

pathologists before enrollment. Biopsies were evaluated and classified according to the

diagnostic criteria [69].

Regarding the interstitial evaluation there was used a semiquantitative score evaluation by

visual rating conducted by two independent pathologists through a scoring system from 0 to 3,

as measured percentage interstitial inflammatory infiltrate and interstitial fibrosis percentage

of total cortical area, there were excluded glomerular and tubular areas. Thus, score 0 = no

inflammatory infiltrates or interstitial fibrosis; score 1 = presence of changes in <25% of

cortical area assessed, score 2 = 25-50% presence of changes in cortical area assessed, score 3

= presence of changes in> 50% of the cortical area assessed.

Interobserver reproductivity was assessed by Spearman's correlation test that varied

between 0.82 and 0.96 and the Student t test for paired data revealed no significant difference

between observers.

The morphometric study was aimed to study histological semiquantitative measurement of

the density of collagen fibers. Tubulo-interstitial area images in trichrome Goldner-Szeckeli

staining were previously processed in Adobe Photoshop before the morphometric study was

conducted in order to remove the overestimation of fibrosis by exclusion of tubular basement

membrane.

Regarding statistical analysis, continuous variables were expressed as mean values ± SD.

Differences between groups were determined by Student t test, Mann-Whitney test and

ANOVA, where indicated. Also, Kaplan-Meier analysis was used to assess survival, statistical

significance was established for P values <0.05. Statistical analysis was performed using

SPSS software version 9.

II.2.2. Results

The most common histopathological diagnoses encountered in the study group were

represented by membranous nephropathy (17.2%), minimal change glomerulopathy (16%)

and focal segmental glomerulosclerosis (11.2%) and in the group that was observed the

decline in renal function the most common forms of pathologic diagnosis was chronic tubulo-

interstitial nephropathy, membranoproliferative glomerulonephritis and renal amyloidosis.

In 13% of cases diagnosed with membranous nephropathy was observed glomerular

capillary walls with weak electron-dense deposits epimembranare and common areas of

resorbtion, glomerular basement membranes on electron microscopy they have characteristics

of stage IV membranous nephropathy .

Of the 8 patients who had renal function decline, respectively initiated renal replacement

therapy, 5 biopsies (62.5%) had renal function decline in more than 12 months after the renal

biopsy puncture. In these biopsies it was observed the overall inflammation score greater than

0 in 80% of cases and a score of inflammation in interstitial fibrosis area greater than 0 in

20% of cases.

In the cases that had a total inflammation score greater than 0 forms were the

predominant histopathological lesions nephropathies with active lesions:

membranoproliferative glomerulonephritis, vascular nephropathy.

In renal biopsies in which inflammation score <1 was observed a lower percentage of

progression to initiation of renal replacement therapy during follow-up, thus, only in one case

was initiated the renal replacement therapy in the follow-up period of the 28 cases of biopsy

who had a total score of inflammation equal to 0 and 2 cases of 50 biopsies who had a score

of inflammation in interstitial fibrosis area equal to 0.

In 64% of cases that showed vascular congestion this was associated with the presence of

total inflammation score greater than 1 (r = 0.29, p = 0.031). It was observed a characteristic

vascular congestion associated with the presence of plasma cell inflammatory lymphocytes in

the interstitial area.

A characteristic noted was the association of periglomerular fibrosis in early stages of

glomerulosclerosis characterized by capillaries with open lumen compared with the presence

of periglomerular fibrosis with well-defined aspect of stratified collagen deposition

surrounding Bowmann capsular basement membrane in a severe glomerulosclerosis scoring.

Average score of tubulointerstitial fibrosis in the interstitial area was 42.3 ± 11.1% for

renal biopsies with interstitial fibrosis confirmed by visual assessment.

In renal biopsies with interstitial fibrosis semiquantitative score 0 the average fibrosis

assessed by computer analysis was 9.1 ± 7.4%. The interstitial fibrosis was observed

prominently as a deposition of fibrous bands at the interstitial and peritubular level and in

some cases this is associated with an inflammatory infiltrate with plasmo-lymfocitary cells.

Thus, there was observed the presence of interstitial fibrosis score higher in the group of

patients that recorded the decline in renal function during the follow-up (r = 0.61, p = 0.03).

Also, interstitial fibrosis score was associated with expression of periglomerular fibrosis (r =

0.21, p = 0.001).

II.2.3. Discussions

In the study group was observed that the most common histological types were

represented by membranous nephropathy, minimal change glomerulopathy and focal

segmental glomerulosclerosis. These data are consistent with the literature, which emphasizes

the high frequency of primary glomerulonephritis and the high prevalence of

histopathological forms of membranoproliferatve glomerulonephritis, focal segmental

glomerulosclerosis and minimal change glomerulopathy [74].

Recent data indicate that lymphoid neogenesis can play an important role in maintaining

immune responses and thus, promote chronic inflammation [77].

Thus, tissues housing a chronic inflammatory process express a high density of effectors

cells such as T lymphocytes, macrophages, dendritic cells, B lymphocytes which can be

organized as secondary lymphoid follicles that can be structured in lymphatic nodes and

lymphatic T cells areas [78,79].

The impact of inflammation on renal function reflects association with progressive

disease, presenting histopathological markers of inflammation expressed both in areas with

interstitial fibrosis and no fibrosis areas, supporting the assertion that renal dysfunction is not

primary caused by the independent inflammatory process [80]. Thus, severe injury to the

nephron causes an inflammatory reaction allowing the nephron and stromal remodeling as an

attempt to healing tissue.

It was observed the presence of tubular atrophy, interstitial fibrosis in severe degree in the

group that recorded the decline in glomerular filtration rate over a period of time greater than

one year from the moment of renal biopsy. Thus, in these biopsies the association of

histopathological markers of inflammation in areas with or without fibrosis, interstitial

fibrosis, inflammatory reaction is caused due to loss of nephron, this explains why

inflammation markers correlates with the decline of renal function [88].

Various studies as the study by Remuzzi et al. revealed that single sections correctly

estimated advanced glomerulosclerosis but overestimated by about 30% the percentage of

normal glomeruli [89]. Thus, in this study there are limitations imposed by histopathological

evaluation of a small number of sections in the correct classification of individual

glomerulosclerosis score for each section.

Regarding periglomerular fibrosis in our study was observed the association between

expression of periglomerular fibrosis and glomerulosclerosis score. These results are

consistent with data presented in various studies that emphasizes the importance of assessing

periglomerular fibrosis in routine histopathological analysis of renal biopsies [90].

Multiple studies have demonstrated the presence of non- reversible healing mechanisms as

a key pathogenic factor in the development of chronic kidney disease regardless of initial

histological lesions [92]. Thus, chronic injury to the parenchyma, similar to well differentiated

tissue structures replacement by fibrosis in the healing process, are specific pathogenetic

mechanisms in the development of chronic renal parenchymal insufficiency of tissues such as

liver, kidneys and lungs [93].

II.3. IMMUNOHISTOCHEMICAL STUDY

Immunohistochemical study objectives were represented by: epithelial phenotype

characteristics observing the histopathological forms of chronic lesions in the renal biopsy,

the description of the immunohistochemical expression of epithelial and mezemchymal

markers in tubulo-interstitial, evaluation of the importance of tubulo-interstitial markers

assesment in the evaluation of chronic kidney disease.

II.3.1. Matherials and methods

The immunohistochemical study was conducted on a total of 36 renal biopsies selected

following exclusion criteria, from the initial population of 87 pieces of renal biopsy, obtained

from patients who underwent renal biopsy procedures between April 2008 - September 2011

and followed for a period of 17 ± 11 months or until the initiation of renal replacement

therapy or death.

An important exclusion criterion in the study was the absence of immunohistochemical

biopsy material, which was used in many cases almost entirely for diagnostic histopathology.

Another exclusion criterion was the histopathological forms of acute kidney injury or acute

proliferative lesions such as proliferative diabetic nephropathy, acute interstitial nephritis,

acute tubular necrosis, vasculitis or thrombotic microangiopathy.

To highlight the immunohistochemical method used is two-staged tissue antigens based

on a polymer network visualization system (DAKO EnVision).

Similar to the histological the immunohistochemically stained slides were then

investigated using a Nikon Eclipse microscope (Nikon, Apidrag, Romania) equipped with a

5MP CCD objectives and an AxioCam MRc5 digital vidocamera.

Quantitative analysis was performed using imaging morphometric analysis dedicated

software Image Pro Plus (MediaCybernetics) [68]. Areas of interest were photographed with

the same lighting and contrast settings and the images were stored in TIF format in a database.

Immunohistochemical study was conducted on a database of more than 200 microscopic

images captured with 20x and 40x objectives.

Antibodies used in the immunohistochemical study are presented below:

Tabel 1. Antibodies used in the immunohistochemical study

Antibody Marker Dilution Antigen retrieveal Source

α- SMA

CD68

Cytokeratine

(MNF116)

E-cadherin

Vimentin

Ki-67

Myofibroblasts

Macrophages/monocites

Cytokeratines with

intermediate molecular

weight

Epithelial adesion

molecule

Mezemchymal marker

Proliferation marker

1:50

1:50

1:50

1:500

1:150

1:50

Citrat buffer, pH6

Citrat buffer, pH6

Citrat buffer, pH6

Citrat buffer, pH 6

Citrat buffer, pH6

EDTA, pH 9

Dako

Dako

Dako

Epitomics

Invitrogen

Dako

The morphometric study aimed to measure the intensity and percentage of area occupied

by the signal, the number of items of interest identified and the distance between the elements

of interest.

Has only been studied aspects tubulo-interstitial area were excluded from morphometric

analysis of glomerular areas or structures in the vicinity of large vessels.

The expression of immunohistochemical markers was expressed as a percentage of

cortical tubulointerstitial area intestiţială, busy signal, exception made CD-68 expression

being represented by the number of positive cells / interstitial area analyzed.

Similar histological study was used a semiquantitative scores to quantify tubulointerstitial

lesions in the interstitial area represented by diagnostic scores ranging between 0 and 3, level

chronic glomerular lesions, vascular, tubular or interstitial [90] .

Regarding statistical analysis, continuous variables were expressed as mean values ± SD.

Differences between groups were determined by Student t test, Mann-Whitney test and

ANOVA, where indicated. Also, Kaplan-Meier analysis was used to assess survival in the

study group, the primary endpoint being the serum cretininei increase by more than 25% from

baseline at the time of renal biopsy (T0), observed during the tracking. Statistical analysis was

performed using SPSS software 9.

II.3.2. Results

It has been observed the male predominance in the group of patients included in the

immunohistochemical study, similar to the original population. In this group was not observed

any case where there has been encountered the decline in glomerular filtration rate with the

initiation of renal replacement therapy during follow-up.

Also it was observed during follow-up in 63.8% of cases serum creatinine increase during

follow-up with more than 25% compared to the value recorded at the renal biopsy moment.

Thus, it was observed in the study group the classification according to the stage of chronic

kidney disease, the presence of 25% of cases in stage III chronic kidney disease, 38.8% of

stage II cases of chronic kidney disease and 41.6% in stage I chronic kidney disease at the

time of renal biopsy.

The main forms of histopathological diagnosis were represented by focal segmental

glomerulosclerosis in 27.7% of cases, followed by membranous nephropathy in 22.2% of

cases and diabetic nephropathy in 16.6% of cases.

In the main histopathological features in the population for immunohistochemical study,

there was a percentage of glomerulosclerosis expression by 14 ± 4.2%, in this study group no

relationship was observed between the expression score of glomerulosclerosis and renal

function parameters evolution.

It was also observed the expression of interstitial fibrosis score with an average of 31.2 ±

16.1%. Another aspect observed was the expression of interstitial fibrosis of 11.1 ± 3.5% in

the expression of inflammatory infiltrate with a score> 1, compared with the expression of

interstitial fibrosis of 23.1 ± 8.2% in the absence of inflammatory infiltrate (p = 0.03).

Morphometric expression of interstitial fibrosis was associated with development of renal

function parameters. Thus, in cases in which there has been observed the increase in serum

creatinine during follow-up, showed a higher expression of interstitial fibrosis with a mean of

54 ± 11.7% compared with the group that was not observed the increase in serum creatinine

during follow-up (p = 0.01).

In this study, cytokeratines expression was assessed as the percentage expression observed

in tubular sections for each biopsy, cytokeratines and E-cadherin expression was not observed

in interstitial area, also, no expression was observed at the glomerular epithelial area.

An important aspect observed in this study was the decreased expression of cytokeratines

expression in biopsies with interstitial fibrosis semiquantitative score greater than 1.

Thus, cytokeratines expression was observed with an average of 31.8 ± 5.9% of tubular

sections. It has also been observed in this study a decrease in the expression of cytokeratines

in 34% of renal biopsies who had interstitial fibrosis score greater than 1 (r = -0.61, p = 0.03).

In the study group were observed association of low expression of tubular cytokeratines

epithelium expression with predominant expression of myofibroblasts (r = -0.32, p = 0.02)

and vimentin (r = -0.25, p = 0.002) in the tubulo-interstitial area.

Similar to expression of cytokeratines it was evaluated the E-cadherin expression which is

a transmembrane adhesion molecule important in defining the tubular epithelial phenotype

characteristics, this marker was not observed in interstitial or glomerular area.

The E-cadherin expression was observed in the tubular epithelium with an average of 37.1

± 11% of tubular sections. Also observed in the group who had increased serum creatinine

during follow-up E-cadherin expression was with an average of 24.5 ± 11.5%, lower than the

expression of E-cadherin in the group that showed no increase in serum creatinine during

follow-up (p = 0.02).

Vimentin expression in the tubulo-interstitial area was associated with the interstitial

fibrosis and also it was observed the immunohistochemical presence of vimentin in tubular

epithelial cells.

In the evolution of renal function parameters was observed in the group that did not show

tubular epithelial expression of vimentin association with a glomerular filtration rate

calculated by the MDRD formula with an average of 43.8 ± 14.5 mil/min/1.73m ² higher than

group showing vimentin expression in tubular epithelium (p = 0.002).

In this study, it was not observed the myofibroblasts expression in tubular epithelial cells

but mainly at interstitial area and also was observed at the glomerular area. Interstitial

expression of myofibroblasts are mainly in the areas of interstitial fibrosis or in the adjacent

areas with vimentin expression.

There was no relationship between immunohistochemical expression of interstitial

myofibroblasts and other parameters of renal function evaluation such as proteinuria/24h or

glomerular filtration rate at renal biopsy moment.

Also in this study it was observed the relationship between myofibroblasts expression and

serum creatinine variation during follow-up. Thus, there is a negative association between

immunohistochemical expression of α-SMA and changes in serum creatinine during follow-

up (r = -0.23, p = 0.01).

Imnunohistochemical expression of macrophages was not observed in the glomerular

capillaries and tubular epithelial cells. Immunoexpression of interstitial CD-68 was between

0.4 and 11.2 positive cells / field.

In the group of patients who presented increased serum creatinine during follow-up, there

was the expression of a larger number of CD-68 positive cells with a mean of 5.5 ± 3.2

positive cells compared with the group did not had increased creatinine during follow-up (p =

0.04).

An important aspect observed in this study was the association of a immunoexpression of

CD-68 interstitial positive cells higher in the patients who were not treated with vitamin D

analogues or substitutes with an average of 6.4 ± 2.1 cells / field compared to the group

receiving vitamin D therapy (p = 0.02).

There was not observed the presence of proliferation markers in tubular epithelial cells at

interstitial level.

In the multivariate analysis found no relationship between expression of myofibroblasts

and vimentin and evaluated clinical parameters such as age, male gender and the presence of

several cardiovascular risk factors.

However, analyzing the vimentin expression in renal function predictivity evolution, only

the immunohistochemical markers demonstrated a diagnostic specificity and sensitivity

statistically significant even at low levels of expression, in the prediction of serum creatinine

increase during the follow-up (AUC 0.680, p = 0.0026).

II.3.3. Discussions

Tubular expression of vimentin was observed to be important for prognosis of renal

function progression when its expression is associated with interstitial infiltration of

myofibroblasts.

In this study, vimentin expression was associated with the degree of renal dysfunction at

the biopsy moment, but also is a strong predictor of the development of renal function

parameters during follow-up.

In addition, the presence of tubular vimentin expression showed an association with

inflammatory infiltrate, indicating that the inflammatory events and those related with EMT

are closely related and possibly represent different stages in the same sequence pathogenic

outcome in the progressive deterioration of renal function [95].

According to these results, a previous study reported an association between changes in

phenotype tubular interstitial area, the degree of interstitial fibrosis and renal function

parameters expression, respectively, serum creatinine [96].

However, it should be noted that, although it has been observed in various studies, the

association between EMT and chronic tubulo-interstitial fibrosis and chronic deterioration of

renal function have not yet found evidence to support a causal role of EMT in decline in

glomerular filtration rate.

Also, interstitial expression of myofibroblasts are demonstrated as a predictor of

development of renal function in renal biopsy protocol performed in less than 12 months post-

transplant biopsies [94].

It is important to note that the association between vimentin expression and

miofibroblaştilor on the one hand, and the evolution of renal function on the other hand, was

independent of serum creatinine or renal biopsy proteinurei/24h at the time. Based on these

results, it is clear that a wider use of these markers may represent an important element in the

development of renal prognosis.

Another important aspect described in the immunohistochemical study was represented of

interstitial macrophages immunoexpression evaluation, this expression is associated with

progression of chronic lesions in the renal biopsy.

Multiple studies have described glomerular or interstitial nephropathies as characterized

by the accumulation of macrophages in interstitial area, this process can become chronic and

further stimulate the production of growth factors similar to the healing process [99,100].

The association between tubular expression of vimentin and myofibroblasts infiltration in

the immunohistochemical study has not been demonstrated to be a process of epithelial to

mesenchymal transition (EMT), respectively, in the analysis of histological sections was not

observed the tubular basement membrane discontinuation.

Although in this study was not investigated the interstitial myofibroblasts origin, the

association of myofibroblasts infiltration and macrophages in the areas of early fibrosis calls

for possible dual origin of these cells or fibroblasts at local or systemic origin of the cells

derived from bone marrow

These observations are consistent with the study by Yu et al. denying the presence of a

epithelial to mesenchymal transdifferentiation process at tubulo-interstitial renal in the

fibrosis development process [102].

IV.CONCLUSIONS

1. In terms of population distribution by gender was observed both in the clinical

and statistical study as well as for immunohistochemical study, an increased

incidence of males in certain age groups. After the age of 60 years was observed

predominance of female patients in renal biopsy indication.

2. In the clinical study, conducted in a statistical group of patients with different

etiology of renal impairment was observed that performing renal biopsy at a

glomerular filtration rate greater was associated with improved survival or fewer

cases in which was observed the initiation of renal replacement therapy.

3. In patients with diabetes and proteinuria/24h> 3g revealed a small number of

cases with indication to perform renal biopsy, included in the study versus those

with altered renal function, eGFR calculated by the MDRD formula

<90ml/min/1.73m ² and nephrotic proteinuria.

4. In analyzing the factors involved in the development of renal pathology an

important aspect was the predictive power of serum creatinine when renal biospiei

and decreased glomerular filtration rate during follow-up was a predictive factor

for the initiation of renal replacement therapy.

5. The clinical-statistical study also described the importance of treatment with

vitamin D, both at the biopsy moment and during follow-up. A negative

association was observed between biological inflammatory syndrome and vitamin

D treatment, but no improvement was observed in the development of chronic

kidney disease in the vitamin D treated group.

6. The impact of inflammation on renal function reflects association with

progressive disease, histopathological showing inflammation markers expressed

both in areas with interstitial fibrosis and no fibrosis areas, is supporting the

observation that the decline of renal function is due not dependently of the

inflammatory process.

7. Interstitial fibrosis coefficient determined by morphometric study represent a

predictive factor for decline in renal function.

8. Regarding periglomerular fibrosis it has been observed the association between

expression of periglomerular fibrosis and glomerulosclerosis.

9. Also, immunohistochemical study confirmed the importance of assessing

interstitial inflammatory infiltration in the early phase of the development of

interstitial fibrosis process.

10. Another important aspect that was described in the immunohistochemical

study was represented of the interstitial macrophages immunoexpression

evaluation, this expression is associated with progression of chronic lesions in the

renal biopsy.

11. Tubular expression of vimentin was observed to be important for prognosis of

progression for renal function when its expression is associated with interstitial

infiltration of myofibroblasts.

12. In this study, vimentin expression was correlated with the degree of renal

dysfunction at the biopsy moment, but also is a strong predictor of the

development of renal function during follow-up.

13. In the immunohistochemical study, α-SMA expression was not found in

tubular epithelial cells, but only in the interstitial compartment in areas with a

higher degree of tubulo-interstitial fibrosis, collagen deposition and tubular

epithelial cells expressing vimentin.

14. It is important to note that the association between vimentin expression and

myofibroblasts on the one hand, and the evolution of renal function on the other

hand, was independent of serum creatinine or proteinuria/24h.

15. An important aspect observed in the immunohistochemical study was the

association between interstitial macrophage infiltration and treatment with vitamin

D or vitamin D analogs

16. An original aspect described in this study is the association between the

expression of histological epithelial and mesenchymal markers describing the

relationship in the development of tubulo-interstitial fibrosis in the evaluation of

renal function.

17. Immunohistochemical expression of epithelial and mesenchymal markers in

the tubulo-interstitial area, are important prognostic factors in the evolution of

renal function.

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